Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Stimulation of antibody formation by bacterial lipopolysaccharide. The influence of repeated doses of lipopolysaccharide on antibody formation

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   Липополисахарид стимулирует образование антител даже при 8-кратном введении в сочетании с частично очищенным дифтерийным анатоксином.     
   
   

IL-4 is required for the IgE and IgG1 increase and IgG1 autoantibody formation in mice treated with mercuric chloride

Previous studies have established that in susceptible mouse strains, such as A.SW (H-2s), repeated injections of subtoxic doses of HgCl2 induce increased serum levels of total IgE and IgG1, high serum titers of antinuclear autoantibodies (ANo1A), and immune-complex glomerulonephritis. Moreover, it has been shown that susceptibility is determined by H-2As and that Th cells are required for the induction of these immunopathologic alterations by HgCl2. In the present study we showed that treatment in vivo with anti-IL-4 mAb completely abrogated the HgCl2-induced increase in total IgE and partially inhibited the increase in IgG1, but failed to suppress the increase in IgG2A. Furthermore, we showed that IL-4 influences the pattern of IgG subclass distribution among ANo1A of HgCl2-treated mice. Whereas treatment with anti-IL-4 mAb significantly reduced the titers of IgG1 ANolA, it increased those of IgG2A, IgG2B, and IgG3 ANolA. Thus, these results show that IL-4 contributes to the optimal formation in vivo of murine IgG1 and that it is involved in the autoantibody formation of a systemic autoimmune disease. The available evidence suggests that HgCl2 induces an increased production of IL-4 by Th2 cells. If this is correct, it implies that MHC class II alleles determine whether the preferential response to HgCl2 is made by Th1 or Th2 cells and, hence, the type of immunopathologic alterations ensuing.     

Melittin-specific monoclonal and polyclonal IgE and IgG1 antibodies from mice

Melittin, a bee venom peptide consisting of 26 amino acid residues, elicited high IgG and IgE antibody responses in mice of BALB/c and CAF1 strains, but not in mice of A/J, AKR, and C57BL/6 strains. Greater than 80% of the melittin-specific antibodies in sera of responder mice were found to bind the hydrophilic carboxyl-terminal heptapeptide of melittin. Three melittin-specific monoclonal antibodies were obtained from responder mice by the hybridoma technique. Two are of the IgG1 isotype and one is of the IgE isotype. One monoclonal antibody of the IgG1 isotype binds the carboxyl-terminal heptapeptide of melittin, while the other two monoclonal antibodies do not. However, competitive binding studies suggest that all three monoclonal Ig bind at the same, or adjacent, site of melittin. These findings, together with the known amphiphilic property of melittin, suggest that the immunogenicity of this peptide is a consequence of its binding to cell surface phospholipids.     

Immunologic and physicochemical properties of enhancing soluble factors for IgG and IgE antibody responses.

Rabbits were immunized with dinitrophenyl-coupled Ascaris antigen (DNP-Asc) or ragweed antigen (DNP-Rag) included in aluminum hydroxide gel and their mesenteric lymph node cells were cultured for 24 hr in vitro in the presence of free homologous carrier. The cell-free supernatant thus obtained enhanced both IgG and IgE antihapten antibody responses of DNP-primed cells to DNP-heterologous carrier conjugate (DNP-keyhole limpet hemocyanin). Since the cell-free supernatant obtained from Rag-specific cells enhanced antibody response of hapten-primed cells raised by immunization with DNP-Asc, no carrier specificity was involved in the enhancement. It was found that treatment of primed cells with 10-5 M pactamycin suppressed the formation of the enhancing soluble factor, whereas the factor was readily formed in the presence of 2 mug/mol of cytosine arabinoside in the culture. The results indicated that cell proliferation was not required but de novo synthesis of protein was essential for the formation of soluble factor(s). The enhancing factor was not absorbed by either carrier-coated or anti-carrier antibody-coated immunosorbent. It was also found that the enhancing factor was formed by incubating primed cells with carrier-coated Sepharose. The cell-free supernatant containing no free carrier enhanced both IgG and IgE anti-hapten antibody responses. The activities of the cell-free supernatant to enhance IgG and IgE antibody responses were not absorbed by anti-Fab, anti-gamma-or anti-mu-chain antibody immunosorbent, indicating that the nonspecific enhancing factor did not possess immunoglobulin determinant. The cell-free supernatant was fractionated by sucrose density gradient ultracentrifugation and by gel filtration with three radiolabeled proteins, i.e., IgG, ovalbumin, and cytochrome C as markers. Enhancing activity for IgG antibody response was recovered in a fraction between ovalbumin peak (40,000 m.w.) and cytochrome C peak (20,000 m.w.). The activity for IgE antibody response was recovered in a fraction containing IgG marker (150,000 m.w.). By block electrophoresis, both activities were detected in beta globulin fraction. The results suggested that different T cell factors are involved in the IgG and IgE antibody responses.     

Reaginic antibody formation in the mouse. VII. Induction of suppressor T cells for IgE and IgG antibody responses.

Intravenous injections of urea-denatured ovalbumin (UD-OA) into OA-primed high responder mice suppressed the antibody response not only to the priming antigen but also to subsequent immunization with dinitrophenyl derivatives of OA (DNP-OA). The transfer of normal spleen cells or OA-primed spleen cells into UD-OA-treated animals did not restore the capacity of responding to DNP-OA to form anti-DNP IgE and IgG antibodies. The transfer of splenic T cell fraction from the UD-OA-treated animals into normal syngeneic mice diminished both IgE and IgG antibody responses of the recipients to DNP-OA. The B cell-rich fraction from the same donors failed to affect the anti-hapten antibody response and enhanced anti-cancer (OA) IgG antibody response of the recipients. It was also found that the transfer of T cell-rich fraction of OA-primed spleen cells failed to suppress antibody response of the recipients to DNP-OA. The results indicated that spleen cells of UD-OA-treated mice contained suppressor T cells which are distinct from helper cells. Suppressive activity of T cells in the UD-OA treated animals was specific for OA. The transfer of the T cell-rich fraction failed to suppress anti-DNP antibody response of the recipients to DNP-KLH.     

Adjuvant properties of bacterial product cantastim

Aluminum compounds have been used as adjuvants in practical vaccination for more than 60 years to induce an early, an efficient and a long lasting protective immunity. Nowadays they are the most widely used adjuvants in both veterinary and human vaccines. Unfortunately these adjuvants do not only cause undesirable side effects, but often induce T-helper type 2 (Th2)-biased responses. In this study we investigated the ability of the bacterial product CANTASTIM (CS) to augment the immune responses to a model antigen, tetanus toxoid (TT). Immunization of mice with TT+CS elicited higher anti-TT IgG antibody levels as compared to mice that received TT alone. Moreover, treatment with TT+CS resulted in a lower IgG1/IgG2a ratio and a stronger in vitro IFN-gamma synthesis by splenocytes and T cells cocultured with macrophages. These data suggest that CS can be used to enhance the magnitude of the immune response and to skew it towards the Th1 type.     

Specific IgE and IgG4 antibodies to Japanese cedar pollen and total IgE antibody in lumbermen

Serum levels of specific IgE and IgG4 antibodies to Japanese cedar (Cryptomeria japonica) pollen and total IgE antibody in 75 lumbermen and in 53 male office workers at an urban establishment were measured by means of an enzyme linked immunosorbent assay (ELISA) and compared. No significant differences of specific IgE and IgG4 to cedar pollen and total IgE were found between the lumbermen and the office workers. There were no significant differences of incidence of cedar pollinosis and positive (greater than 100 FU/ml) rate of serum specific IgE between the two groups, though the lumbermen were exposed to dense concentrations of cedar pollen in their working area. In the lumbermen who showed positive values of specific IgE, the mean value of the specific antibody in Japanese cedar pollinosis lumbermen was significantly higher than that in symptom-free lumbermen, while no significant differences of serum level of specific IgG4 were found between the two groups.     

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide

IL-4 promotes IgG1 and IgE secretion by murine B cells stimulated with bacterial LPS. We show that stimulation of unprimed resting splenic B cells with LPS and 10(4) U/ml rIL-4 results in the expression of membrane (m) IgG1 and mIgE on 40 to 50% and 15 to 25% of the total B cell population, respectively, on day 4 of culture. The possibility of a significant contribution to cell surface staining by cytophilic, secreted Ig isotypes was eliminated by either the addition of anti-Fc gamma or anti-Fc epsilon R mAb during the culture or by acid treatment before staining. A similar proportion of IgE-expressing B cells are also found, after stimulation with LPS and 10(4) U/ml IL-4, by cytoplasmic staining using fluorescence microscopy. Cell sorting analysis further indicates that B cell populations that express mIgG1 and mIgE secrete these respective Ig isotypes. In addition, such cells show striking diminution in IgM secretion compared to mIgG1- or mIgE- sorted B cells. Stimulation with LPS and IL-4 (10(4) U/ml) induces co-expression of mIgG1 and mIgE on LPS-stimulated B cells; up to 75% of mIgE+ B cells co-express mIgG1 and up to 19% of mIgG1+ B cells express mIgE. This striking co-expression of mIgG1 and mIgE is mirrored by the co-expression of mIgG1 with mIgG3 and mIgG2b by B cells stimulated with LPS and 200 U/ml IL-4. Cell sorting analysis demonstrates that the B cell population that co-expresses mIgG1 and mIgE secretes both IgG1 and IgE. However, "two-color" cytoplasmic staining fails to demonstrate any B cells that simultaneously secrete both IgG1 and IgE.     

Cholesterol inclusion in liposomes affects induction of antigen-specific IgG and IgE antibody production in mice by a surface-linked liposomal antigen

In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.     

Capacity for IgE antibody production in ASK mice

The capacity for IgE anti body production in ASK mice, which are highly sensitivity to anaphylactic shock, was compared with that in C3H and AKR mice. Three strains of mice were immunized with DNP-Ascaris mixed with aluminum hydroxide gel. IgE antibody to DNP in the sera was titrated by the rat 48-hour passive cutaneous anaphylaxis test. Maximum IgE titers of each strain of mice were 1: 2560 in C3H, 1: 1280 in ASK and 1: 640 in AKR. IgE antibody was detected in the sera until 170 days in C3H, 290 days in ASK and for not less than 320 days in AKR. These results suggest that the ASK mouse is a high responder strain for IgE antibody.